The long range goal of this proposal is to define in detail the protein associations of isoforms of erythrocyte membrane structural proteins, spectrin and ankyrin, that have been discovered to be general components of cell plasma membranes, and to elucidate cellular functions of these protein linkages. The approach will be to analyse quantitatively the intermolecular associations of purified, radiolabeled brain spectrin, brain ankyrin, and defined proteolytic domains of these proteins, with proteins in membrane and cytoplasmic fractions. These measurements will a) provide assays for identification and purification of binding proteins; b) allow evaluation of regulation of the protein interactions; c) be used to screen for toxins and mutations that perturb specific protein associations. Specific goals are in the following areas: (1) studies of protein associations of brain spectrin and defined domains of brain spectrin with proteins in membranes and the cytosol. Newly identified spectrin-binding proteins will be purified and characterized. Regulation of spectrin-protein association will be studied with Ca++, calmodulin, and various protein kinases. (2) Brain ankyrin. This protein will be purified and characterized regarding modification by phosphorylation/dephosphorylation and its cellular localization. (3) Protein associations of brain ankyrin will be defined and binding proteins purified as described with brain spectrin. (4) Functions of integral membrane proteins that bind to ankyrin. (5) Cellular functions of protein associations described above will be evaluated by a) using defined protein domains or antibody against against these domains as competitive antagonists of protein associations in living cells, b) screening toxins for modulation of protein associations with the goal of identifying a specific inhibitor, c) screening mutant mice for alteration in function or expression of spectrin, ankyrin or any binding proteins.